Human respiratory syncytial virus (HRSV) is the leading viral cause of serious pediatric respiratory tract disease worldwide (Collins, et al., Fields Virology 2:1313-1352, 1996; incorporated herein by reference). RSV outranks all other microbial pathogens as a cause of pneumonia and bronchiolitis in infants under one year of age. Virtually all children are infected by two years of age, and reinfection occurs with appreciable frequency in older children and young adults (Chanock et al., in Viral Infections of Humans, 3rd ed., A. S. Evans, ed., Plenum Press, N.Y., 1989; incorporated herein by reference). RSV is responsible for more than one in five pediatric hospital admissions due to respiratory tract disease, and in the United States alone causes nearly 100,000 hospitalizations and 4,500 deaths yearly. (Heilman, J. Infect. Dis. 161:402-6, 1990; incorporated herein by reference). In addition, there is evidence that serious respiratory tract infection early in life can initiate or exacerbate asthma (Sigurs, et al., Pediatrics 95:500-505, 1995; incorporated herein by reference).
While RSV usually is thought of in the context of the pediatric population, it also is recognized as an important agent of serious disease in the elderly (Falsey et al., J. Infect. Dis. 172:389-394, 1995; incorporated herein by reference). RSV also causes life-threatening disease in certain immunocompromised individuals, such as bone marrow transplant recipients (Fouillard, et al., Bone Marrow Transplant 9:97-100, 1992; incorporated herein by reference).
For treatment of RSV, one chemotherapeutic agent, ribavirin, is available. However, its efficacy and use are controversial. There are also licensed products for RSV intervention which are composed of pooled donor IgG (Groothuis et al., N. Engl. J. Med. 329:1524-1530, 1993; incorporated herein by reference) or a humanized RSV-specific monoclonal antibody. These are administered as passive immunoprophylaxis agents to high-risk individuals. While these products are useful, their high cost and other factors, such as lack of long-term effectiveness, make them inappropriate for widespread use. Other disadvantages include the possibility of transmitting blood-borne viruses and the difficulty and expense in preparation and storage. Moreover, the history of the control of infectious diseases, and especially diseases of viral origin, indicates the primary importance of vaccines.
Despite decades of investigation to develop effective vaccine agents against RSV, no safe and effective vaccine has yet been approved to prevent the severe morbidity and significant mortality associated with RSV infection. Failure to develop successful vaccines relates in part to the fact that small infants have diminished serum and secretory antibody responses to RSV antigens. Thus, these individuals suffer more severe infections from RSV, whereas cumulative immunity appears to protect older children and adults against more serious impacts of the virus.
The mechanisms of immunity in RSV infection have recently come into focus. Secretory antibodies appear to be most important in protecting the upper respiratory tract, whereas high levels of serum antibodies are thought to have a major role in resistance to RSV infection in the lower respiratory tract. RSV-specific cytotoxic T cells, another effector arm of induced immunity, are also important in resolving an RSV infection. However, while this latter effector can be augmented by prior immunization to yield increased resistance to virus challenge, the effect is short-lived. The F and G surface glycoproteins are the two major protective antigens of RSV, and are the only two RSV proteins which have been shown to induce RSV neutralizing antibodies and long term resistance to challenge (Collins et al., Fields Virology, Fields et al. eds., 2:1313-1352, Lippincott-Raven, Philadelphia, 1996; Connors et al., J. Virol. 65 (3):1634-1637, 1991; incorporated herein by reference). The third RSV surface protein, SH, did not induce RSV-neutralizing antibodies or significant resistance to RSV challenge.
An obstacle to developing live RSV vaccines is the difficulty in achieving an appropriate balance between attenuation and immunogenicity. Other obstacles include the genetic instability of some attenuated viruses, the relatively poor growth of RSV in cell culture, and the instability of the virus particle. In addition the immunity which is induced by natural infection is not fully protective against subsequent infection. A number of factors probably contribute to this, including the relative inefficiency of the immune system in restricting virus infection on the luminal surface of the respiratory tract, the short-lived nature of local mucosal immunity, rapid and extensive virus replication, reduced immune responses in the young due to immunological immaturity, immunosuppression by transplacentally derived maternal serum antibodies, and certain features of the virus such as a high degree of glycosylation of the G protein. Also, as will be described below, RSV exists as two antigenic subgroups A and B, and immunity against one subgroup is of reduced effectiveness against the other.
Although RSV can reinfect multiple times during life, reinfections usually are reduced in severity due to protective immunity induced by prior infection, and thus immunoprophylaxis is feasible. A live-attenuated RSV vaccine would be administered intranasally to initiate a mild immunizing infection. This has the advantage of simplicity and safety compared to a parenteral route. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. It also abrogates the immunosuppressive effects of RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications (Murphy et al., Vaccine 8(5):497-502, 1990; incorporated herein by reference), this has never been observed with a live virus.
A formalin-inactivated virus vaccine was tested against RSV in the mid-1960s, but failed to protect against RSV infection or disease, and in fact exacerbated symptoms during subsequent infection by the virus. (Kim et al., Am. J. Epidemiol., 89:422-434, 1969; Chin et al., Am. J. Epidemiol., 89:449-463, 1969; Kapikian et al., Am. J. Epidemiol., 89:405-421, 1969; incorporated herein by reference).
More recently, vaccine development for RSV has focused on attenuated RSV mutants. Friedewald et al., (J. Amer. Med. Assoc. 204:690-694, 1968; incorporated herein by reference) reported a cold passaged mutant of RSV (cpRSV) which appeared to be sufficiently attenuated to be a candidate vaccine. This mutant exhibited a slightly increased efficiency of growth at 26° C. compared to its wild-type (wt) parental virus, but its replication was neither temperature sensitive nor significantly cold-adapted. The cold-passaged mutant, however, was attenuated for adults. Although satisfactorily attenuated and immunogenic for infants and children who had been previously infected with RSV (i.e., seropositive individuals), the cpRSV mutant retained a low level virulence for the upper respiratory tract of seronegative infants.
Similarly, Gharpure et al., (J. Virol. 3:414-421, 1969; incorporated herein by reference) reported the isolation of temperature sensitive RSV (tsRSV) mutants which also were promising vaccine candidates. One mutant, ts-1, was evaluated extensively in the laboratory and in volunteers. The mutant produced asymptomatic infection in adult volunteers and conferred resistance to challenge with wild-type virus 45 days after immunization. Again, while seropositive infants and children underwent asymptomatic infection, seronegative infants developed signs of rhinitis and other mild symptoms. Furthermore, instability of the ts phenotype was detected. Although virus exhibiting a partial or complete loss of temperature sensitivity represented a small proportion of virus recoverable from vaccinees, it was not associated with signs of disease other than mild rhinitis.
These and other studies revealed that certain cold-passaged and temperature sensitive RSV strains were underattenuated and caused mild symptoms of disease in some vaccinees, particularly seronegative infants, while others were overattenuated and failed to replicate sufficiently to elicit a protective immune response, (Wright et al., Infect. Immun. 37:397-400, 1982; incorporated herein by reference). Moreover, genetic instability of candidate vaccine mutants has resulted in loss of their temperature sensitive phenotype, further hindering development of effective RSV vaccines. See generally, (Hodes et al., Proc. Soc. Exp. Biol. Med. 145:1158-1164, 1974; McIntosh et al., Pediatr. Res. 8:689-696, 1974; and Belshe et al., J. Med. Virol. 3:101-110, 1978; incorporated herein by reference).
As an alternative to live-attenuated RSV vaccines, investigators have also tested subunit vaccine candidates using purified RSV envelope glycoproteins. The glycoproteins induced resistance to RS virus infection in the lungs of cotton rats, (Walsh et al., J. Infect. Dis. 155:1198-1204, 1987; incorporated herein by reference), but the antibodies had very weak neutralizing activity and immunization of rodents with purified subunit vaccine led to disease potentiation (Murphy et al., Vaccine 8:497-502, 1990; incorporated herein by reference).
Recombinant vaccinia virus vaccines which express the F or G envelope glycoprotein have also been explored. These recombinants express RSV glycoproteins which are indistinguishable from the authentic viral counterpart, and rodents infected intradermally with vaccinia-RSV F and G recombinants developed high levels of specific antibodies that neutralized viral infectivity. Indeed, infection of cotton rats with vaccinia-F recombinants stimulated almost complete resistance to replication of RSV in the lower respiratory tract and significant resistance in the upper tract. (Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466, 1986; incorporated herein by reference). However, immunization of chimpanzees with vaccinia-F and -G recombinant provided almost no protection against RSV challenge in the upper respiratory tract (Collins et al., Vaccine 8:164-168, 1990; incorporated herein by reference) and inconsistent protection in the lower respiratory tract (Crowe et al., Vaccine 11:1395-1404, 1993; incorporated herein by reference).
Despite these various efforts to develop an effective RSV vaccine, no licensed vaccine has yet been approved for RSV. The unfulfilled promises of prior approaches underscores a need for new strategies to develop RSV vaccines, and in particular methods for manipulating recombinant RSV to incorporate genetic changes that yield new phenotypic properties in viable, attenuated RSV recombinants. However, manipulation of the genomic RNA of RSV and other non-segmented negative-sense RNA viruses has heretofore proven difficult. Major obstacles in this regard include non-infectivity of naked genomic RNA of these viruses and, in the case of RSV, poor viral growth in tissue culture, lengthy replication cycles, virion instability, a complex genome, and a refractory organization of gene products.
Recombinant DNA technology has made it possible to recover infectious non-segmented negative-stranded RNA viruses from cDNA, to genetically manipulate viral clones to construct novel vaccine candidates, and to rapidly evaluate their level of attenuation and phenotypic stability (for reviews, see Conzelmann, J. Gen. Virol. 77:381-389, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-11358, 1996; incorporated herein by reference). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), parainfluenza virus (PIV), rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), rinderpest virus and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-4481, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell et al., EMBO J. 13:4195-4203, 1994; Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-8392, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997; Pecters et al., J. Virol. 73:5001-5009, 1999; Kato et al., Genes to Cells 1:569-579, 1996; Roberts et al., Virology 247(1), 1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; International Publication No. WO 97/06270; U.S. Provisional Patent Application No. 60/007,083, filed Sep. 27, 1995; U.S. patent application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S. Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996; U.S. Provisional Patent Application No. 60/046,141, filed May 9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999 (corresponding to International Publication No. WO 98/02530); U.S. Provisional Patent Application Ser. No. 60/129,006, filed Apr. 13, 1999; Collins, et al., Proc. Nat. Acad. Sci. USA 92:11563-11567, 1995; Bukreyev, et al., J. Virol. 70:6634-6641, 1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997; Durbin et al., Virology 235:323-332, 1997; He et al. Virology 237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997; Whitehead et al., Virology 247(2):232-239, 1998a; Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; and Whitehead et al., J. Virol. 73:(4)3438-3442, 1999, and Bukreyev, et al., Proc. Nat. Acad. Sci. USA 96:2367-2372, 1999, Bucholz et al., J. Virol. 73:251-259, 1999; Collins et al., Virology 259:251-255, 1999, each incorporated herein by reference in its entirety for all purposes).
Based on these developments in recombinant DNA technology, it is now possible to recover infectious RSV from cDNA and to design and implement various genetic manipulations to RSV clones to construct novel vaccine candidates. Thereafter, the level of attenuation and phenotypic stability, among other desired phenotypic characteristics, can be evaluated.
One avenue of investigation toward developing recombinant vaccines has been to engineer viruses to express one or more cytokines or other potential anti-viral molecules. For the most part, these studies involved wild type vaccinia virus and were conducted with the goal of increasing basic understanding of host immunity and viral pathogenesis (see, e.g., Ramshaw et al. Immunol. Rev. 127:157-182, 1992; incorporated herein by reference). The potential utility of cytokine coexpression to improve immune responses for vaccine development has long been contemplated (Ramshaw et al., Trends Biotechol 10:424-426, 1992; incorporated herein by reference). However, the use of poxvirus as an object of study reduced the practical application of this concept since smallpox has been eradicated in the human population and the poxvirus vaccine no longer remains in active use.
Examples of these earlier studies investigating the possible utility of cytokine coexpression for vaccine development include a vaccinia virus engineered to express the cytokine interleukin 2 (IL-2). This recombinant virus was reported to be attenuated in immunodeficient athymic nude mice (Flexner et al., Nature 330:259-262, 1987; Ramshaw et al., Nature 329:545-546, 1987; incorporated herein by reference). The roles of various immune effectors in clearance and recovery have also been investigated (Karupiah et al., J. Ex. Med. 172:1495-1503, 1990; Karupiah et al., J. Immunol. 144:290-298, 1990; Karupiah et al., J. Immunol. 147:4327-4332, 1991; incorporated herein by reference). IL-2 expression by the vaccinia virus recombinant was shown to greatly reduce skin lesions formed by the vaccinia virus in primates, indicating significant attenuation. Despite these preliminary findings, antibody production was determined to be equivalent in the presence, or absence, of IL-2 (Flexner et al., Vaccine 8:17-21, 1990; incorporated herein by reference).
Another example of cytokine coexpression by a recombinant vaccinia virus involved interleukin 4 (IL-4), which was reported to downregulate antiviral cytokine expression and cytotoxic T cell responses, and to exacerbate the viral infection (Sharma et al., J. Virol. 70:7103-7107, 1996; incorporated herein by reference). In yet another study, expression of nitric oxide synthetase by recombinant vaccinia virus was highly attenuating, demonstrating the importance of this host defense mechanism in controlling vaccinia virus infection (Rolph et al., J. Virol. 70:7678-7685, 1996; incorporated herein by reference). Coexpression of IL-5 or IL-6 by a recombinant vaccinia virus resulted in a 4-fold elevation in the level of local IgA (Ramsay et al., Reprod. Fertil. Dev. 6:389-392, 1994; incorporated herein by reference). Coexpression of tumor necrosis factor (TNF) alpha by a recombinant vaccinia virus improved the capacity of mice to control infection, suggesting that this molecule is involved in host defenses against the virus (Sambhi et al., Proc. Natl. Acad. Sci. USA 88:4025-4029, 1991; incorporated herein by reference). Expression of murine IFNγ by a recombinant vaccinia virus greatly reduced virus replication in normal or immune-compromised mice (Kohonen-Corish, Eur. J. Immunol. 20:157-161, 1990; incorporated herein by reference).
These studies have also been extended to retroviruses. Recently, simian immunodeficiency virus (SUV) expressing IL-2 was constructed (Gundlach et al., J. Virol. 71:2225-2232, 1997; incorporated herein by reference). In rhesus monkeys infected with the IL-2-expressing virus the SIV-specific T-cell proliferative response and the antibody titers were similar to those of control virus. SIV-specific CTL were detected in monkeys infected with the IL-2-expressing virus and in two of four control animals. In another study, a SIV recombinant lacking the nef gene and containing the IFNλ gene was attenuated in vivo, but the cytokine gene was unstable after several weeks of replication in vivo and, in addition, the attenuation was associated with reduced immunogenicity (Giavedoni et al., J. Virol. 71:866-872, 1997; incorporated herein by reference).
Studies with large double-stranded poxviruses, which have approximately 185 ORFs and encode a number of proteins which interfere with host defenses, or with human immunodeficiency virus which also interferes with host defense, are poor models for the nonsegmented negative strand RNA viruses. It has been previously demonstrated that a foreign gene can be expressed from the genome of recombinant respiratory syncytial virus (RSV), and that it is stably maintained (Bukreyev J. Virol. 70: 6634-6641, 1996, incorporated herein by reference). This indicates that it is feasible to coexpress proteins such as cytokines, chemokines, ligands, and other molecules which might alter, increase or otherwise enhance the host response to RSV. The expression of one or more immune modulatory molecules from a recombinant RSV is desirable because it would provide for expression at the local site of RSV antigen production. Furthermore, coexpression obviates the need to separately prepare and administer the immune modulator. However, the strategy of expressing an immune modulator from the genome of a non-retrovirus, i.e., a positive-sense, double-stranded, or negative-sense RNA virus, had not been previously explored.
There is presently a need for augmentation and modification of host immune responses to RSV. This is because an RSV vaccine will be administered to very young infants, an age group which is known to mount immune responses that are less effective and in some respects different than those of adults. For example, neutralizing antibody responses and cytotoxic T cell responses to RSV are reduced in the very young (Kovarik and Siegrist, Immunol. Today 19: 150-152, 1998; Kovarik and Siegrist, Immunol. Cell. Biol. 76:222-236, 1998; Murphy et al., J. Clin. Microbiol. 24:894-989, 1986; Risdon et al., Cell. Immunol. 154:14, 1994; incorporated herein by reference). It is generally recognized that the neonatal immune system is biased towards a Th-2 type T helper cell response (i.e., the T helper cell subset defined by IL-4 secretion) (Early and Reen, Eur. J. Immunol. 26:2885-2889, 1996; incorporated herein by reference). T cells from newborns have reduced helper cell activity for B cells, and produce lower quantities of a number of cytokines, including IL-2, interferon gamma (IFNγ), and IL-4 (Splawski et al., J. Clin. Invest. 87, 454, 1991; Hassan and Reen, Scand. J. Immunol. 39:597, 1994; Wilson, Pediatr. 54:118, 1991; incorporated herein by reference). Also, young infants typically possess transplacentally-derived RSV-specific IgG, which can interfere with or alter immune responses (Murphy et al., J. Clin. Microbiol. 24:894-989, 1986 and 23:1009-1014, 1986; Siegrist et al., Eur. J. Immunol. 28:4138-4148, 1998; incorporated herein by reference). Finally, certain RSV immunizations can be associated with immunopathologic complications (Murphy et al., Vaccine 8:497-502, 1990; Waris et al., J. Virol. 71:6935-6939, 1997; incorporated herein by reference). Although this typically is not observed with live-attenuated virus infections, there is evidence that individual RSV antigens, and most notably the G protein, have the potential to induce immunopathologic responses even when expressed by live virus (Johnson et al., J. Virol. 72:2871-2880; incorporated herein by reference). These many factors involved in RSV immune responses, immune-mediated protection, and immunopathologic responses indicate a need for developing methods to modulate host responses to an RSV vaccine.
In summary, an urgent need presents itself in the art to develop additional tools and methods to engineer safe and effective vaccines that will alleviate the serious health problems attributable to RSV. In this context, it is necessary to develop a broad and diverse menu of genetic modifications that can be employed, alone or in combination with other types of genetic manipulations, to construct infectious, attenuated RSV vaccine candidates useful for broad vaccine use. Useful manipulations to a live attenuated RSV vaccine virus might include coexpression by a recombinant RSV clone of or more one factors that modulate host immune responses and immune-mediated protection. Surprisingly, the present invention fulfills this need by providing additional tools for constructing infectious, attenuated RSV vaccine candidates.